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A Comparison of Respiratory Pathways in Fully Habituated and Normal Non-organogenic Sugarbeet Callus
• JOURNAL OF • PLANT PHYSIOLOGY
J Plant Physiol WJl 156. pp. 312-318 (2000) http://www.urbanfischer.de/journals/jpp
e 2000 URBAN &FISCHER Verlag
A Comparison of Respiratory Pathways in Fully Habituated and Normal Non-organogenic Sugarbeet Callus Badia Bisbis I, Anneke M. Wagner2, Claire Kevers l and Thomas GasparI 1
Institute of Botany B 22, University of Liege - Sart Tilman, B-4000 Liege, Belgium
2
IMHW; Molecular Cell Physiology, Vrije Universiteit, De Boelelaan 1087, NL-lOSl HY. Amsterdam, The Netherlands
Received February 15, 1999 . Accepted July 2, 1999
Summary
The respiration of cells and isolated mitochondria of a habituated non-organogenic (HNO) cell line (auxin- and cytokinin-independent) and of a normal (N) cell line (auxin- and cytokinin-requiring) from sugarbeet (Beta vulgaris L.) was investigated. Oxygen uptake by both cells and mitochondria of the HNO line was higher than that of the N line. Respiration in the presence of cyanide (i.e. capacity of the alternative pathway) was also higher in the HNO callus as compared with the N one. The measurements of O 2 uptake from isolated HNO and N mitochondria showed that addition of a mixture of substrates (NADH, succinate, malate and NAD) resulted in a higher respiration via the CN-resistant pathway in mitochondria from HNO cells. The activity of cytochrome c oxidase was twice as high as in the N cells as compared with the HNO cells. Immunoblots also showed that a higher alternative oxidase protein was present in HNO cells than in the N cells. A model showing the relationships between the different metabolic pathways previously studied in the HNO cells and the higher respiration via the CN-resistant pathway in the HNO cells is proposed.
Key words: Alternative pathway, Beta vulgaris, callus, cytochrome pathway, habituation, mitochondria, sugarbeet. Introduction
Cells from a habituated non-organogenic (HNO) sugarbeet callus show morphological and biochemical traits similar to animal cancer cells (Gaspar et al., 1991; Hagege et al., 1992a; Crevecoeur et al., 1992). Cells from the HNO line exhibit abnormal mitochondrial structures (Crevecoeur et al., 1992) and alterations in the control of the tetrapyrrolic metabolism with a deficiency in cytochromes, catalase and peroxidases (Hagege et al., 1992 b). The HNO cells accumulate higher amounts of polyamines and present a higher GABA-shunt activity (Bisbis et al., 1997 a). In contrast to the green normal (N) callus line from the same plant, the white HNO callus accumulates glucose and fructose with a higher fructose/glucose ratio. The non-photosynthetic cells from the HNO callus are heterotrophic (Bisbis et al., 1994). It has also been shown that the cells from HNO callus evolved less CO 2 and exhibited a higher malate
dehydrogenase activity than cells from the N callus (Carrie et al., 1994, 1995). Cells from the HNO line have a higher oxygen consumption than N callus (Carrie et al., 1995). In plant cells, oxygen is taken up and reduced to water through a mitochondrial electron transport chain. The oxygen uptake may proceed through two pathways: the CN-sensitive cytochrome pathway (CP), and the CN-resistant alternative pathway (AP), which is inhibited by hydroxamates (Lambers, 1985; Larhrissi et al., 1994). The electron transfer via the two pathways is branched at the Q-pool (Storey, 1976) and diverges after complex I (NADH dehydrogenase), the only site of ATP production via the CN-resistant respiration. The AP is a non-phosphorylating pathway bypassing two of the three sites of proton translocation (III and IV); thus, the AP is not coupled to the ATP synthesis. The dehydrogenases reduce ubiquinone to ubiquinol, which is reoxidized by the two pathways (Moore and Siedow, 1991). In addition to the internal dehydrogenases (complex I and a succinate dehydro0176-1617/00/156/312 $ 12.00/0
Respiratory Pathways in Sugarbeet Calli
genase: complex 11), plant mitochondria contain external dehydrogenases that donate electrons to the Qpool: an NADHand an NADPH-dehydrogenase (Arron and Edwards, 1980; M0ller and Lin, 1986; Roberts et al., 1995). The aim of the present work was to elucidate the relative activities of the two respiratory pathways in the HNO callus in comparison to the N callus. The differences in activities of the respiratory pathways is discussed in relation to the metabolic deviations previously described in HNO cells (Le Dily et al., 1993; Bisbis et al., 1997 a, b; Gaspar et al., 1998).
313
O2 uptake measurements Whole tissue Respiration of calli cells (about 1 g FW) was measured at 25 ·C and neutral pH using a YSI oxygen monitor model 53. Different concentrations of BHAM (benzohydroxamate) for the alternative oxidase inhibition (0 to 15 mmollL) and KCN for cytochrome pathway inhibition (0 to 2 mmollL) were used. The total respiration (uninhibited respiration) is defined as the total oxygen uptake in the absence of the inhibitors. The uncoupler, 5-chloro-3-t-buryl-2'chloro-4'-nitrosalicylanilide (S-I3), was supplied at a final concentration of 0.5 IlmollL. Respiration was expressed as Ilmoles O 2 , h- I . (g dry weight)-I.
Materials and Methods
Plant material and culture conditions Experimental conditions for obtaining normal (N) and habituated non-organogenic (HNO) callus of sugarbeet (Beta vulgaris L. var. altissima) and for maintaining these tissues in solid stock cultures under light (16 h ghotoperiod of Sylvania Grolux fluorescent light providing 17W m - , 25 .C) have been reported elsewhere (Kevers et al., 1999). Such calli are subcultured (transfer of 200 to 500 mg callus pieces, in 10 mL plastic Petri dishes) every 2 weeks on their respective solid medium: basal medium without plant growth regulators for the habituated lines, but supplemented with 0.451lmol IL 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.441lmo1/L benzylaminopurine (BAP) for normal lines.
Mitochondria Respiration of isolated mitochondria was measured in 1 mL washing medium (II) at 25 ·C at pH 7.1 using a YSI oxygen monitor model 53. BHAM (2 mmollL) was used to inhibit the alternative pathway and 0.1 mmollL KCN was used to inhibit the cytochrome pathway. The respiration was measured after addition of substrates: NADH (2 mmollL), succinate (20 mmollL) and malate (20 mmoll L), in the presence of2mmollL NAD. State 3 respiration was measured in the presence of 11lmoi/L ADP.
Enzyme assays Growth evaluation and dry weight determination For fresh weight (FW) measurements, callus was removed from the Petri dishes, rapidly blotted, placed on aluminium foil, and weighed. Initial FW was calculated by the difference between Petri dishes with and without callus immediately after subculturing. For dry weight (DW) determination, calli were removed from the medium, weighed for the fresh weight determination and dried at 60 ·C for 24 h.
Isolation of mitochondria Cells (10-30 g fresh weight) of 3-week-old calli were suspended in 100 mL of medium containing 0.4 mollL mannitol, 0.1 mollL phosphate buffer, 1 % (w/v) cellulase (Onozuka R-lO, Serva) and 0.1 % pectinase (from Aspergillus niger, Serva); the pH was adjusted to 5.7. The calli were incubated for 1 h on an orbital shaker (100 rpm) at room temperature. Completion of cell wall digestion was verified microscopically. From this moment, all of the treatments were performed at 4·C. Protoplasts were collected by centrifugation and broken with a mortar and a pestle in a medium containing 0.4 mollL mannitol, 10 mmollL EDTA (ethylenediaminetetraacetate), 2 mmollL L-cysteine, 0.2 % BSA (bovine serum albumin), 0.7 % PVP-25 (polyvinylpyrrolidone) and 1 mmollL phosphate buffer, pH 7.4. The homogenate was centrifuged at 3,000 go for 10 min, the supernatant was centrifuged at 10,000 go for 10 min and the resulting pellet was resuspended in washing medium (I) containing 0.4 mollL mannitol, 0.2 % BSA, 10 mmollL phosphate buffer and 1 mmollL L-cysteine, pH 7.2, and layered on top of a self-generating Percoll gradient. After centrifugation at 30,000 go for 30 min, the mitochondrial band was collected and diluted in a washing medium (II) containing 0.4 mollL mannitol, 0.2 % BSA and 10 mmollL phosphate buffer, pH 7.1, then centrifuged at 10,000 go for 10 min. The resulting pellet was resuspended in about 0.5 mL of washing medium II.
Cytochrome c oxidase (EC. 1.9.3.1) was measured in the mitochondrial suspension obtained as described above. The mitochondrial suspension was diluted 10 times in 10 mmol/L phosphate buffer, pH 7.4, supplemented with 0.1 % (w/v) Triton X-100 and centrifuged at I3,000 go for 2 min. The supernatants were used for spectrophotometrical assay. Cytochrome oxidase was measured by the decrease of absorbance at 550 nm in the presence of 0.03 mmollL reduced cytochrome c in 0.1 mollL potassium phosphate buffer (pH 7.2). Cytochrome c (from horse heart, Boehringer Mannheim) was reduced with sodium dithionite and the excess of sodium dithionite was removed by applying the cytochrome clsodium dithionite mixture to a Dowex column, type 1 X 8, mesh width 100-200 and eluted with distilled water. Activities were calculated as the first order constant k (mgprotein)-I. min-I. Peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities were assayed as described in Bisbis et aI. (1997b, c). Glucose-6-phosphate dehydrogenase (G6PDH, EC. 1.11.149) and the ATP-dependent phosphofructokinase (PFK, EC. 2.7.1.1 1) activities were assayed as described in Bisbis et al. (1993). Mitochondrial and total protein was determined by the Bradford method (Bradford, 1976) using BSA for calibration.
SDS-PAGE and immunoblotting One hundred Ilg of mitochondrial protein was solubilized in sample buffer (Tris [312 mmollL, pH 6.8], 10 % SDS [w/v], 10 % glycerol (v/v) , 0.002 % bromophenol blue and 100 mmollL DTT) and boiled for 1-2 min. The mitochondrial samples were subjected to SDS-polyacrylamide electrophoresis (10 %), followed by Western blotting. Antibodies developed against the alternative oxidase protein of Sauromatum guttatum (generously supplied by Dr. T. Elthon) were used at dilutions of 1/1000. Visualization was with the ECL chemiluminescent reagent system (Arnersham) as described in Wagner (1995).
314
Badia Bisbis, Anneke M. Wagner, Claire Kevers and Thomas Gaspar
Results
The whole tissue respiration experiments indicated that total respiration (uninhibited) was higher in the HNO cell line (Fig. 3 A), which is in agreement with previous results from Carrie et al. (1995). The respiration in the presence of BHAM was not different in the two types of calli during the culture period as shown in Fig. 3 B. However, the O 2 consumption rate in the presence of cyanide was higher in HNO callus during the culture period as compared with N callus (Fig.3C). The respiration rates, measured in the presence ofKCN and BHAM, represent the maximal rates that can be measured in whole tissue for the cytochrome and the alternative pathways, respectively. Respiratory rates can be controlled by: 1) the energy status, especially control of ADP/ATP ratios on glycolysis and/or mitochondrial respiratory chains, and 2) enzyme capacities of respiratory pathways. In order to test the first possibility, the respiration rates of the two types of callus were also measured in the presence of the uncoupler S 13. The uncoupler induced an increase in the uninhibited and cytochromal respiration rates in both types of callus, but the uncoupled respiration rates increased more in N callus than in HNO callus (Table 1). This suggested that the ADP/ATP ratios control N callus respiration to a higher extent. Even in the presence of the uncoupler, uninhibited respiration remained higher in HNO callus than in N callus, suggesting that differences in enzyme capacities playa role, too.
The growth rate of the habituated non-organogenic callus is lower than that of the normal callus (Fig. 1). The growth curve of HNO callus is characterized by a shorter linear growth phase compared with the N callus. In order to investigate whether a change in respiratory characteristics in the two types of callus could be responsible for the differences in growth rate, total cell respiration and respiration in the presence ofBHAM (CP) and KCN (AP) were determined. The concentrations of BHAM and KCN required to obtain a maximum inhibition of the AP and CP in sugarbeet cells were determined through the use of titration curves. The final concentration of KCN required for a maximum inhibition of CP ranged from 0.4 to 1 mmol/L (Fig. 2A). The final concentration of BHAM required for maximum inhibition of the AP ranged from 5 to 20 mmol/L (Fig. 2 B). Titration with BHAM in the presence of 1 mmol/L KCN showed that some residual O 2 consumption (insensitive to the inhibitors) was present in the two types of calli (Fig. 2 C). Concentrations of 1 mmol/L KCN and 10 mmol/L BHAM were used in the experiments described below. 700 -'"""'
~
600
"
500
~
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]
~ ~
400 300 200
~
100 0
0
---
14
7
Table 1: Respiration rates of 3-day-old N and HNO calli in the presence and in the absence of the uncoupler (S13). Increased respiratory rates are expressed as % of the control.
--I
Respiration rates [J.lffiol O 2 . h -1 • (g OW) -1]
21
-513
28
,-,
~
300
r
e
200
-
100
'-
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%
+CN 309.0±15.4 469.7±14.9 340.0±15.4 110 458.4±28.1 97 382.2±32.6 428.3±21.5 450.9±19.6 118 456.3±16.7 106 +BHAM +CN+BHAM 144.2±12.3 107.3± 18.0 187.0±12.9 129 134.1±17.8 125
400
:.:.,;
C!l
%
405.0±23.9 589.3±34.5 535.6±34.2 132 658.7±42.6 112
Uninhibited
Fig. 1: Growth rates of the N (e) and the HNO (.) calli. The growth indexes are expressed as relative fresh weight (FW) increase: [(final FW-initial FW)/initial FW] X 100. Results (± SD) are means of three separate experiments.
N
HNO
N
Time of culture (days)
+S13
t: 0
-
A
l
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B
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1
1
2
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CN concentrations (mM)
f-I
o
1
5
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~ ~ o
C
1
I
c\t:!=! -I
10 IS 20 2S 0 BHAM concentrations (mM)
5
10
IS
20
25
BHAM concentrations (mM)
Fig. 2: Respiration (Ilmoles O 2 • h-1 • g (DW)-l) of 21-day-old N (e) and HNO (.) calli in the presence of different concentrations of KCN (A) or BHAM (B) or BHAM in the presence of 1 mmol/L ofKCN (C). Results (± SD) are means of three separate measurements.
Respiratory Pathways in Sugarbeet Calli
600
Table 2: Respiratory rates of isolated mitochondria from Nand HNO cells after 21 days of culture.
A
500
nmol O 2 , min-I. (mg prot)-l
400
Uninhibited
300
-~ Q
00
'-'
-:.c:
rJi
~ 0
e::1.
§ .::: c..
~
§ to>
0 '"
315
HNO
N
200
eN-resistant N
BHAM-resistant
HNO
N
HNO
Mixture of 89.8±7.3 159.7± 14.6 63.2±9.6 152.6±21.0 88.5±5.6 89.9±8.7
100
substrates
0 500
B
400
Table 3: Enzyme activities of the N and the HNO calli after 21 days of culture. N
300 200 100 0 500
~
400 300 200
c
\ \ \-
100
---
Glucose-6-phosphate dehydrogenase (J.!g NADPH.min-l.mg prot-I) Phosphofructokinase (J.!g NADH.min-l.mg proC I) cytochrome c oxidase (k.min-I.mg prot-I) Peroxidase (nkat. mg proC I) Catalase (J.!kat.mg proC I) Total protein content (mg.gDW- I)
HNO
B.50±O.1l
1B.OO±O.04
2.70±O.OB
1.40±O.O2
10.42±O.O2
5.35±O.46
5.B3±O.54
O.47±O.O6
1.43±O.O2
l.O4±O.O4
50.0±U
103.9±2.5
- . - --:t:
0 0
7 14 21 28 Time of culture (days)
Fig. 3: Uninhibited respiration (A) (J.lmoles O 2 ·h-I . g (DW)-I) ofN (e) and HNO (_) calli and respiration in the presence of BHAM (B) and CN (C). Results (± SD) are means of three separate experiments.
In order to measure the electron transport chain capacities, experiments with isolated mitochondria were performed. In isolated mitochondria, when using a mixture of substrates, a similar pattern was observed as in the whole tissue respiration: higher uninhibited respiration, a similar cytochrome pathway and a higher CN-resistant pathway in HNO callus as compared with N callus (Table 2). Table 3 shows some enzyme activities, not only of the respiratory chains (cytochrome c oxidase), but also of several other pathways such as the pentose phosphate pathway (glucose-6-phosphate dehydrogenase), glycolysis (phosphofructokinase), and activities of peroxidase and catalase involved in H 20 2 detoxification. The results showed a higher G6PDH activity and a lower PFK activity in HNO cells as compared with the N cells. Peroxidase and catalase showed also lower activities in HNO cells (Table 3). Cytochrome c oxidase is two times lower in HNO cells than in N cells. Western blotting (in the absence of reducing agents like DTT) of mitochondrial proteins of Nand HNO calli pre-
~.I.It.~ 'I"
N
HNO
Fig. 4: Amount of alternative protein in mitochondria isolated from N and HNO calli, determined by Western blotting. One hundred J.!g protein per lane for both N and HNO cells. sented a band at about 35 kda protein, which corresponds to the alternative oxidase in its reduced form (Fig. 4). Much more alternative oxidase protein appeared to be present in HNO mitochondria compared with N mitochondria.
Discussion
Respiratory characteristics Total respiration and CN-resistant respiration were much higher in HNO callus than in N callus, but BHAM-resistant, cytochromal respiration was the same in the two types of cal-
316
Badia Bisbis, Anneke M. Wagner, Claire Kevers and Thomas Gaspar [ Glycolysis
I..
PFK
Glueose-6-phosph.k
_G6iiiiiiiiDiiiiHi004~ Pentose phosphate .......
Pentosea
pathway
p-r-----~r=~--+_t----------_.srn~~~~_t~--r=====~ co,
~
PEP
"
----t~~
Pyruvate
~m
nK
OAA
tMDH
MAL
Cytosol
"1°1
accumUla.on Lack in catalase and ~roxidase _-::...-"", ~ Lack in ~ tetrapyrroli com ounds
Fig. 5: Scheme illustrating the relationships between the deviated carbon and nitrogen metabolisms and the induction of the alternative oxidase in HNO cells. CIT, citrate; DAO, diamine oxidase; DH, dehydrogenase; e-, electron flux; GABA, y-aminobutyrate; GABA-T, GABA transaminase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; G6DH, glucose-6-phosphate dehydrogenase, GLU, glutamate; KG, a-Ketoglutarate; MAL, malate; MDH, malate dehydrogenase; OM, oxaloacetate; PAO, polyamine oxidase; Pas, polyamines; PEp, phosphoenolpyruvate; PEPC, PEP carboxylase; PFK, phosphofructokinase; SSDH, succinic semialdehyde dehydrogenase; SUC, succinate.
Ius. HNO callus showed a higher CN-resistant respiration than BHAM-resistant respiration. The two pathways coexisted also in N callus but with a low uptake via the CNresistant pathway as compared with the cytochrome pathway. However, it is not possible to determine exact contributions of the two pathways on the basis of these inhibitors studies (Wagner and Krab, 1995). The results presented here would suggest that the difference observed in uninhibited respiration between the two types of callus was mainly due to a difference in the respiration via the CN-resistant pathway. Accordingly, the respiration rates of isolated mitochondria with a mixture of exogenous substrates (succinate, malate and NADH) were higher in mitochondria from HNO cells, with a higher respiration rate via the CNresistant pathway and no difference in BHAM-resistant cytochrome respiration between the N and the HNO callus. The higher respiration rates in HNO callus could be caused by a difference in overall enzyme capacities or a difference in ADP/ATP ratio, controlling glycolytic flux. If cellular respiration is ADP availability dependent, a strong stimulation of respiration by uncoupler can be expected. Indeed, an increase of total and cytochrome mediated respiration was observed in the presence of the uncoupler. This indicates that the cytochrome pathway is not working at full capacity. Still, uncoupled respiration in HNO cells was higher than in N
cells, indicating that enzyme capacities were higher in the HNO cell line. More alternative oxidase protein (35 kda) was present in mitochondria isolated from HNO callus compared with N callus mitochondria. This is in agreement with a higher capacity of this pathway. The activity of the alternative pathway can be regulated by the total amount of protein, but also by the reduction state of the enzyme (Umbach and Siedow, 1996). Cytochrome c oxidase activities, on the contrary, were higher in N callus than in HNO callus, which apparently does not affect cytochromal respiratory rates. Similar results were found by Van Emmerik et al. (1993). The higher glucose-6-phosphate dehydrogenase and the lower phosphofructokinase activities in HNO callus point to a disturbed sugar metabolism preferentially deviated to the pentose phosphate pathway. This results probably in a lower glycolytic pathway of HNO callus with, as consequence, an atypical tricarboxylic acid cycle, which is replenished by anaplerotic pathways such as malate production by the CO 2 fixation via phosphoenolpyruvate carboxylase and succinate production via the aminobutyrate shunt (see Fig. 5) (Bisbis et al., 1997a; Gaspar et al., 1998). It is not clear whether the lower peroxidase and catalase activities in HNO callus resulted in less H 2 0 2 elimination in this callus compared with the N callus. Further investigations on that H 20 2 accumulation in HNO callus must be performed.
Respiratory Pathways in Sugarbeet Calli
Respiratory pathways and growth The HNO callus presents a lower growth and higher respiratory rate, the contrary is true for the N callus. The maximal cytochromal activities of the two rypes of callus are the same; thus, the ATP synthesis could be expected to be similar. Recent work has shown that the HNO callus presented a higher level of ATP compared with its normal counterpart (Poder et al., 1998). The differences in growth rates of the HNO and N cells cannot then be explained only by the differences in respiratory characteristics. An explanation for the lower growth of the HNO callus could be found in the hormonal status and in several disturbed metabolic pathways already studied (Gaspar et al., 1998). Indeed, the HNO callus is characterized by a higher endogenous auxin than its normal counterpart (Bisbis, 1998), a lower ethylene production (Bisbis et al., 1998), and a lower auxin oxidase activiry (Kevers et al., 1999). On the other hand, we have shown that although the HNO callus contains no more chlorophyll and is nonphotosynthetic as compared with the N callus (Bisbis et al., 1994), still about 20 % of the biomass increase derives from CO 2 fixation versus 44 % for the N callus (Bisbis et al., 1997 a). The CO 2 fixation occurred in HNO callus via the phosphoenolpyruvate carboxylase (Bisbis et al., 1997 a). The HNO callus presents a deficiency in tetrapyrrolic compounds, such as peroxidase and catalase (Bisbis et al., 1998). Some or all of these parameters could contribute to the lower growth with a reduced post-exponential linear growth phase ofHNO, which could indicate a defective cell expansion (Hagege et al.,
1991).
Conclusions
O 2 uptake by HNO cells is higher than in N cells and is more CN-resistant than respiration in N cells. The alternative oxidase protein was shown to be present in a higher amount in HNO callus than in N callus. The HNO callus was a subject of many studies, which showed many metabolic deviations and suggested that the HNO callus is under permanent stress (Gaspar et al., 1998 and refs. therein). In this respect, the observed increase of alternative oxidase protein is interesting. Plants are able to regulate, in a coordinate fashion, the partitioning of electrons between the cytochrome and the alternative pathways to meet changing metabolic requirements (Vanlerberghe and McIntosh, 1992). Little is known, however, about the functions of the alternative oxidase (AP). Alternative oxidase is induced in response to stress conditions and it has been suggested that the alternative oxidase plays a role in the antioxidant defence in plants (Purvis and Shewfelt, 1993; Wagner and Krab, 1995; Wagner and Moore,
1997).
A reaction to the lowered cytochrome c oxidase activiry shown in HNO cells might be the synthesis of extra-alternative oxidase protein, as observed before (Vanlerberghe and McIntosh, 1992; Wagner and Wagner, 1997). It is not clear yet how the signal from the cytochrome pathway to nuclear gene expression is transduced, but reactive oxygen species such as H 20 2 can act as inducers (Wagner, 1995). It is well known that HNO callus synthesizes a higher amount of poly-
317
amines (Hagege et al., 1990) yielding H 20 2 via their degradation. The lower catalase and peroxidase activities could facilitate the accumulation of H 20 2 • An increased alternative oxidase is in accordance with this assumption. Figure 5 summarizes the different metabolic pathways investigated in HNO callus. Acknowledgements
B. Bisbis would like to thank the FNRS (Fonds National de la Recherche Scientifique) for supporting this work, and the scientific and technical staff of IMBW, Molecular Cell Physiology Laboratory.
References
Arron, G. P., and G. E. Edwards: Oxidation of reduced nicotinamide adenine nucleotide phosphate by potato mitochondria. Plant Physiol. 65,591-594 (1980). Bisbis, B., F. Le Dily, C. Kevers, J. P. Billard, C. Huault and Th. Gaspar: Disturbed sugar metabolism in a fully habituated nonorganogenic callus of Beta vulgaris L. Plant Growth Regul. 11, 257-261 (1993). Bisbis, B., E. Dujardin, C. Kevers, D. Hagege and Th. Gaspar: Chlorophylls and carotenoids in a fully habituated non-organogenic callus of Beta vulgaris. BioI. Plant. 36, 443-449 (1994). Bisbis, B., C. Kevers and Th. Gaspar: Atypical TCA cycle and replenishment in a non-photosynthetic fully habituated sugarbeet callus overproducing polyamines. Plant Physiol. Biochem. 35, 363-368 (1997a). Bisbis, B., C. Kevers, C. Huault, J. P. Billard and Th. Gaspar: Erythromycin as a tool to investigate the tetrapyrrole biosynthetic pathway in habituated and normal sugarbeet calli. Plant Growth Regul. 23, 147-152 (1997b). Bisbis, B., C. Kevers, C. Pene!, H. Greppin and Th. Gaspar: Coexistence of the Beale and the Shemin pathways for the tetrapyrrolecontaining compounds' synthesis, including peroxidases, in a normal sugarbeet callus. In: Plant Peroxidase Newsletter, Univ. of Geneva 9,13-20 (1997c). Bisbis, B.: Auxin, ethylene and polyamine interactions in a habituated sugarbeet callus. Bull. Soc. Roy. Sc. Liege 67, 213 (1998). Bisbis, 8., C. Kevers, J. Creche, M. Rideau and Th. Gaspar: Differential growth dependency of normal and habituated sugarbeet cell lines upon endogenous ethylene production and exogenous ethylene application. Physiol. Plant. 103,201-208 (1998). Bradford, M. M.: A rapid method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72,248-254 (1976). Carrie, B., 8. Bisbis, C. Pene!, Th. Gaspar and H. Greppin: Disturbed sugar metabolism in a fully habituated non-organogenic callus of Beta vulgaris. Additional data. Saussurea 25, 143-151 (1995). Carrie, 8., Th. Gaspar, H. Greppin and C. Pene!: Redox characteristics of normal and habituated cell lines of sugarbeet. Plant Cell Environ. 17, 457-461 (1994). Crevecoeur, M., D. Hagege, A. M. Catesson, H. Greppin and Th. Gaspar: Ultrastructural characteristics of cells from normal and habituated sugarbeet calli. Plant Physiol. Biochem. 30, 87-95 (1992). Gaspar, Th., 8. Bisbis, C. Kevers, C. Pene!, H. Greppin, F. Le Dily, J.P. Billard, C. Huault, F. Garnier, M. Rideau and J. M. Foidart: Atypical metabolisms and biochemical cycles imposing the cancerous state on plant cells. Plant Growth Regul. 24, 135-144 (1998).
318
Badia Bisbis, Anneke M. Wagner, Claire Kevers and Thomas Gaspar
Gaspar, Th., D. Hagege, c. Kevers, C. Penel, M. Crevecoeur, I. Engelmann, H. Greppin and]. M Foidart: When plant teratomas turn into cancers in the absence of pathogens. Physiol. Plant. 83, 696-670 (1991). Hagege, D., R. Catania, H. Micalef and Th. Gaspar: Nuclear shape and DNA content of fully habituated non-organogenic sugarbeet cells. Protoplasma 166, 49-54 (1992 a). Hagege, D., D. Werck-Reichhart, P. Schmitt and Th. Gaspar: Deficiency in tetrapyrrole-containing compounds in a non-organogenic habituated sugarbeet cell line. Plant Physiol. Biochem. 30, 649-654 (1992 b). Hagege, D., C. Kevers, Th. Gaspar and T. A. Thorpe: Abnormal growth of habituated sugarbeet callus and cell suspension. In Vitro Cell Dev. BioI. 27, 112-116 (1991). Hagege, D., C. Kevers, ]. Boucaud, M. Duyme and Th. Gaspar: Polyamines, phospholipids and peroxides in normal and habituated sugarbeet calli.]. Plant Physiol. 136, 641-645 (1990). Kevers, c., B. Bisbis, O. Faivre-Rampant and Th. Gaspar: Putrescine metabolism in a fully habituated nonorganogenic sugarbeet callus and its relationship with growth. ]. Plant Physiol. 154, 503-508 (1999). Lambers, H.: Respiration in intact plants and tissues: its regulation and dependence on environmental factors, metabolism and invaded organisms. In: Douce, R., and D. A. Day (eds.): Encyclopedia of Plant Physiology, New Series, vol. 18, pp. 418-465. Springer Verlag, New York (1985). Larhrissi, Z., ]. L. Bomsel and R. Calvayrac: Relative effect of 2A-dinitrophenol on the respiratory chain of heterotrophic Euglena gracilis adapted and non-adapted to antimycin. ]. Plant Physiol. 143,264-268 (1994). Le Dily, E,]. P. Billard, Th. Gaspar and C. Huault: Disturbed nitrogen metabolism associated with the hyperhydric status of fully habituated callus of sugarbeet. Physiol. Plant. 88, 129-134 (1993). M0ller, I. M., and W. Lin: Membrane-bound NAD(P)H dehydrogenases in higher plant cells. Annu. Rev. Plant Physiol. 37, 309334 (1986). Moore, A. L., and]. N. Siedow: The regulation and nature of the cyanide-resistant alternative oxidase of plant mitochondria. Biochim. Biophys. Acta 1059, 121-140 (1991).
Poder, D., O. Omarzad and D. Hagege: Comparative study of respiratory metabolism in three rypes of sugarbeet calli, a normal and two habituated ones (organogenic and non-organogenic). C. R. Acad. Sci. Paris, Sciences de la vie, 321, 479-483 (1998). Purvis, A. c., and R. L. Shewfelt: Does the alternative pathway ameliorate chilling injury in sensitive plant tissues? Physiol. Plant. 88,712-718 (1993). Roberts, T. M., K. M. Fredlund and I. M. M0ller: Direct evidence for the presence of two external NAD(P)H dehydrogenases coupled to the electron transport chain in plant mitochondria. FEBS Lett. 373, 307-309 (1995). Storey, B. T.: Respiratory chain of plant mitochondria. XVIII. Point of interaction of the alternative oxidase with the respiratory chain. Plant Physiol. 58, 521-525 (1976). Umbach, A. L., and ]. N. Siedow: The reaction of soybean coryledon mitochondrial cyanide-resistant oxidase with sulfhydryl reagent suggests that a-ketoacid activation involves the formation of a thiohemiacetal. ]. BioI. Chern. 271, 25019-25026 (1996). Vanlerberghe, G. c., and L. Mcintosh: Coordinate regulation of cytochrome and alternative pathway respiration in tobacco. Plant Physiol. 100, 1846-1851 (1992). Van Emmerik, W. A. M., A. M. Wagner,]. H. Zwiers and L. H. W. Van Der Plas: The regulation of respiration in cell suspension of Petunia hybrid4, studied by inhibiting mitochondrial protein synthesis with chloramphenicol. Physiol. Plant. 88,251-258 (1993). Wagner, A. M.: A role for active oxygen species as second messengers in the induction of alternative oxidase gene expression in Petunia hybrid4 cells. FEBS Lett. 368, 339-342 (1995). Wagner, A. M., and K. Krab: The alternative pathway in plants: role and regulation. Physiol. Plant. 95, 318-325 (1995). Wagner, A. M., and A. L. Moore: Structure and function of the plant alternative oxidase: its putative role in the oxygen defence mechanism. Biosci. Rep. 17, 319-333 (1997). Wagner, A. M., and M. ]. Wagner: Changes in mitochondrial respiratory chain components of Petunia cells during culture in the presence of antimycin A. Plant Physiol. 115,617-622 (1997).
J Plant Physiol WJl 156. pp. 312-318 (2000) http://www.urbanfischer.de/journals/jpp
e 2000 URBAN &FISCHER Verlag
A Comparison of Respiratory Pathways in Fully Habituated and Normal Non-organogenic Sugarbeet Callus Badia Bisbis I, Anneke M. Wagner2, Claire Kevers l and Thomas GasparI 1
Institute of Botany B 22, University of Liege - Sart Tilman, B-4000 Liege, Belgium
2
IMHW; Molecular Cell Physiology, Vrije Universiteit, De Boelelaan 1087, NL-lOSl HY. Amsterdam, The Netherlands
Received February 15, 1999 . Accepted July 2, 1999
Summary
The respiration of cells and isolated mitochondria of a habituated non-organogenic (HNO) cell line (auxin- and cytokinin-independent) and of a normal (N) cell line (auxin- and cytokinin-requiring) from sugarbeet (Beta vulgaris L.) was investigated. Oxygen uptake by both cells and mitochondria of the HNO line was higher than that of the N line. Respiration in the presence of cyanide (i.e. capacity of the alternative pathway) was also higher in the HNO callus as compared with the N one. The measurements of O 2 uptake from isolated HNO and N mitochondria showed that addition of a mixture of substrates (NADH, succinate, malate and NAD) resulted in a higher respiration via the CN-resistant pathway in mitochondria from HNO cells. The activity of cytochrome c oxidase was twice as high as in the N cells as compared with the HNO cells. Immunoblots also showed that a higher alternative oxidase protein was present in HNO cells than in the N cells. A model showing the relationships between the different metabolic pathways previously studied in the HNO cells and the higher respiration via the CN-resistant pathway in the HNO cells is proposed.
Key words: Alternative pathway, Beta vulgaris, callus, cytochrome pathway, habituation, mitochondria, sugarbeet. Introduction
Cells from a habituated non-organogenic (HNO) sugarbeet callus show morphological and biochemical traits similar to animal cancer cells (Gaspar et al., 1991; Hagege et al., 1992a; Crevecoeur et al., 1992). Cells from the HNO line exhibit abnormal mitochondrial structures (Crevecoeur et al., 1992) and alterations in the control of the tetrapyrrolic metabolism with a deficiency in cytochromes, catalase and peroxidases (Hagege et al., 1992 b). The HNO cells accumulate higher amounts of polyamines and present a higher GABA-shunt activity (Bisbis et al., 1997 a). In contrast to the green normal (N) callus line from the same plant, the white HNO callus accumulates glucose and fructose with a higher fructose/glucose ratio. The non-photosynthetic cells from the HNO callus are heterotrophic (Bisbis et al., 1994). It has also been shown that the cells from HNO callus evolved less CO 2 and exhibited a higher malate
dehydrogenase activity than cells from the N callus (Carrie et al., 1994, 1995). Cells from the HNO line have a higher oxygen consumption than N callus (Carrie et al., 1995). In plant cells, oxygen is taken up and reduced to water through a mitochondrial electron transport chain. The oxygen uptake may proceed through two pathways: the CN-sensitive cytochrome pathway (CP), and the CN-resistant alternative pathway (AP), which is inhibited by hydroxamates (Lambers, 1985; Larhrissi et al., 1994). The electron transfer via the two pathways is branched at the Q-pool (Storey, 1976) and diverges after complex I (NADH dehydrogenase), the only site of ATP production via the CN-resistant respiration. The AP is a non-phosphorylating pathway bypassing two of the three sites of proton translocation (III and IV); thus, the AP is not coupled to the ATP synthesis. The dehydrogenases reduce ubiquinone to ubiquinol, which is reoxidized by the two pathways (Moore and Siedow, 1991). In addition to the internal dehydrogenases (complex I and a succinate dehydro0176-1617/00/156/312 $ 12.00/0
Respiratory Pathways in Sugarbeet Calli
genase: complex 11), plant mitochondria contain external dehydrogenases that donate electrons to the Qpool: an NADHand an NADPH-dehydrogenase (Arron and Edwards, 1980; M0ller and Lin, 1986; Roberts et al., 1995). The aim of the present work was to elucidate the relative activities of the two respiratory pathways in the HNO callus in comparison to the N callus. The differences in activities of the respiratory pathways is discussed in relation to the metabolic deviations previously described in HNO cells (Le Dily et al., 1993; Bisbis et al., 1997 a, b; Gaspar et al., 1998).
313
O2 uptake measurements Whole tissue Respiration of calli cells (about 1 g FW) was measured at 25 ·C and neutral pH using a YSI oxygen monitor model 53. Different concentrations of BHAM (benzohydroxamate) for the alternative oxidase inhibition (0 to 15 mmollL) and KCN for cytochrome pathway inhibition (0 to 2 mmollL) were used. The total respiration (uninhibited respiration) is defined as the total oxygen uptake in the absence of the inhibitors. The uncoupler, 5-chloro-3-t-buryl-2'chloro-4'-nitrosalicylanilide (S-I3), was supplied at a final concentration of 0.5 IlmollL. Respiration was expressed as Ilmoles O 2 , h- I . (g dry weight)-I.
Materials and Methods
Plant material and culture conditions Experimental conditions for obtaining normal (N) and habituated non-organogenic (HNO) callus of sugarbeet (Beta vulgaris L. var. altissima) and for maintaining these tissues in solid stock cultures under light (16 h ghotoperiod of Sylvania Grolux fluorescent light providing 17W m - , 25 .C) have been reported elsewhere (Kevers et al., 1999). Such calli are subcultured (transfer of 200 to 500 mg callus pieces, in 10 mL plastic Petri dishes) every 2 weeks on their respective solid medium: basal medium without plant growth regulators for the habituated lines, but supplemented with 0.451lmol IL 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.441lmo1/L benzylaminopurine (BAP) for normal lines.
Mitochondria Respiration of isolated mitochondria was measured in 1 mL washing medium (II) at 25 ·C at pH 7.1 using a YSI oxygen monitor model 53. BHAM (2 mmollL) was used to inhibit the alternative pathway and 0.1 mmollL KCN was used to inhibit the cytochrome pathway. The respiration was measured after addition of substrates: NADH (2 mmollL), succinate (20 mmollL) and malate (20 mmoll L), in the presence of2mmollL NAD. State 3 respiration was measured in the presence of 11lmoi/L ADP.
Enzyme assays Growth evaluation and dry weight determination For fresh weight (FW) measurements, callus was removed from the Petri dishes, rapidly blotted, placed on aluminium foil, and weighed. Initial FW was calculated by the difference between Petri dishes with and without callus immediately after subculturing. For dry weight (DW) determination, calli were removed from the medium, weighed for the fresh weight determination and dried at 60 ·C for 24 h.
Isolation of mitochondria Cells (10-30 g fresh weight) of 3-week-old calli were suspended in 100 mL of medium containing 0.4 mollL mannitol, 0.1 mollL phosphate buffer, 1 % (w/v) cellulase (Onozuka R-lO, Serva) and 0.1 % pectinase (from Aspergillus niger, Serva); the pH was adjusted to 5.7. The calli were incubated for 1 h on an orbital shaker (100 rpm) at room temperature. Completion of cell wall digestion was verified microscopically. From this moment, all of the treatments were performed at 4·C. Protoplasts were collected by centrifugation and broken with a mortar and a pestle in a medium containing 0.4 mollL mannitol, 10 mmollL EDTA (ethylenediaminetetraacetate), 2 mmollL L-cysteine, 0.2 % BSA (bovine serum albumin), 0.7 % PVP-25 (polyvinylpyrrolidone) and 1 mmollL phosphate buffer, pH 7.4. The homogenate was centrifuged at 3,000 go for 10 min, the supernatant was centrifuged at 10,000 go for 10 min and the resulting pellet was resuspended in washing medium (I) containing 0.4 mollL mannitol, 0.2 % BSA, 10 mmollL phosphate buffer and 1 mmollL L-cysteine, pH 7.2, and layered on top of a self-generating Percoll gradient. After centrifugation at 30,000 go for 30 min, the mitochondrial band was collected and diluted in a washing medium (II) containing 0.4 mollL mannitol, 0.2 % BSA and 10 mmollL phosphate buffer, pH 7.1, then centrifuged at 10,000 go for 10 min. The resulting pellet was resuspended in about 0.5 mL of washing medium II.
Cytochrome c oxidase (EC. 1.9.3.1) was measured in the mitochondrial suspension obtained as described above. The mitochondrial suspension was diluted 10 times in 10 mmol/L phosphate buffer, pH 7.4, supplemented with 0.1 % (w/v) Triton X-100 and centrifuged at I3,000 go for 2 min. The supernatants were used for spectrophotometrical assay. Cytochrome oxidase was measured by the decrease of absorbance at 550 nm in the presence of 0.03 mmollL reduced cytochrome c in 0.1 mollL potassium phosphate buffer (pH 7.2). Cytochrome c (from horse heart, Boehringer Mannheim) was reduced with sodium dithionite and the excess of sodium dithionite was removed by applying the cytochrome clsodium dithionite mixture to a Dowex column, type 1 X 8, mesh width 100-200 and eluted with distilled water. Activities were calculated as the first order constant k (mgprotein)-I. min-I. Peroxidase (EC. 1.11.1.7) and catalase (EC. 1.11.1.6) activities were assayed as described in Bisbis et aI. (1997b, c). Glucose-6-phosphate dehydrogenase (G6PDH, EC. 1.11.149) and the ATP-dependent phosphofructokinase (PFK, EC. 2.7.1.1 1) activities were assayed as described in Bisbis et al. (1993). Mitochondrial and total protein was determined by the Bradford method (Bradford, 1976) using BSA for calibration.
SDS-PAGE and immunoblotting One hundred Ilg of mitochondrial protein was solubilized in sample buffer (Tris [312 mmollL, pH 6.8], 10 % SDS [w/v], 10 % glycerol (v/v) , 0.002 % bromophenol blue and 100 mmollL DTT) and boiled for 1-2 min. The mitochondrial samples were subjected to SDS-polyacrylamide electrophoresis (10 %), followed by Western blotting. Antibodies developed against the alternative oxidase protein of Sauromatum guttatum (generously supplied by Dr. T. Elthon) were used at dilutions of 1/1000. Visualization was with the ECL chemiluminescent reagent system (Arnersham) as described in Wagner (1995).
314
Badia Bisbis, Anneke M. Wagner, Claire Kevers and Thomas Gaspar
Results
The whole tissue respiration experiments indicated that total respiration (uninhibited) was higher in the HNO cell line (Fig. 3 A), which is in agreement with previous results from Carrie et al. (1995). The respiration in the presence of BHAM was not different in the two types of calli during the culture period as shown in Fig. 3 B. However, the O 2 consumption rate in the presence of cyanide was higher in HNO callus during the culture period as compared with N callus (Fig.3C). The respiration rates, measured in the presence ofKCN and BHAM, represent the maximal rates that can be measured in whole tissue for the cytochrome and the alternative pathways, respectively. Respiratory rates can be controlled by: 1) the energy status, especially control of ADP/ATP ratios on glycolysis and/or mitochondrial respiratory chains, and 2) enzyme capacities of respiratory pathways. In order to test the first possibility, the respiration rates of the two types of callus were also measured in the presence of the uncoupler S 13. The uncoupler induced an increase in the uninhibited and cytochromal respiration rates in both types of callus, but the uncoupled respiration rates increased more in N callus than in HNO callus (Table 1). This suggested that the ADP/ATP ratios control N callus respiration to a higher extent. Even in the presence of the uncoupler, uninhibited respiration remained higher in HNO callus than in N callus, suggesting that differences in enzyme capacities playa role, too.
The growth rate of the habituated non-organogenic callus is lower than that of the normal callus (Fig. 1). The growth curve of HNO callus is characterized by a shorter linear growth phase compared with the N callus. In order to investigate whether a change in respiratory characteristics in the two types of callus could be responsible for the differences in growth rate, total cell respiration and respiration in the presence ofBHAM (CP) and KCN (AP) were determined. The concentrations of BHAM and KCN required to obtain a maximum inhibition of the AP and CP in sugarbeet cells were determined through the use of titration curves. The final concentration of KCN required for a maximum inhibition of CP ranged from 0.4 to 1 mmol/L (Fig. 2A). The final concentration of BHAM required for maximum inhibition of the AP ranged from 5 to 20 mmol/L (Fig. 2 B). Titration with BHAM in the presence of 1 mmol/L KCN showed that some residual O 2 consumption (insensitive to the inhibitors) was present in the two types of calli (Fig. 2 C). Concentrations of 1 mmol/L KCN and 10 mmol/L BHAM were used in the experiments described below. 700 -'"""'
~
600
"
500
~
'-" ~
]
~ ~
400 300 200
~
100 0
0
---
14
7
Table 1: Respiration rates of 3-day-old N and HNO calli in the presence and in the absence of the uncoupler (S13). Increased respiratory rates are expressed as % of the control.
--I
Respiration rates [J.lffiol O 2 . h -1 • (g OW) -1]
21
-513
28
,-,
~
300
r
e
200
-
100
'-
" '0
,;!;
"
t::s
HNO
%
+CN 309.0±15.4 469.7±14.9 340.0±15.4 110 458.4±28.1 97 382.2±32.6 428.3±21.5 450.9±19.6 118 456.3±16.7 106 +BHAM +CN+BHAM 144.2±12.3 107.3± 18.0 187.0±12.9 129 134.1±17.8 125
400
:.:.,;
C!l
%
405.0±23.9 589.3±34.5 535.6±34.2 132 658.7±42.6 112
Uninhibited
Fig. 1: Growth rates of the N (e) and the HNO (.) calli. The growth indexes are expressed as relative fresh weight (FW) increase: [(final FW-initial FW)/initial FW] X 100. Results (± SD) are means of three separate experiments.
N
HNO
N
Time of culture (days)
+S13
t: 0
-
A
l
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'!
B
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1
1
1
2
\
CN concentrations (mM)
f-I
o
1
5
~I \
~ ~ o
C
1
I
c\t:!=! -I
10 IS 20 2S 0 BHAM concentrations (mM)
5
10
IS
20
25
BHAM concentrations (mM)
Fig. 2: Respiration (Ilmoles O 2 • h-1 • g (DW)-l) of 21-day-old N (e) and HNO (.) calli in the presence of different concentrations of KCN (A) or BHAM (B) or BHAM in the presence of 1 mmol/L ofKCN (C). Results (± SD) are means of three separate measurements.
Respiratory Pathways in Sugarbeet Calli
600
Table 2: Respiratory rates of isolated mitochondria from Nand HNO cells after 21 days of culture.
A
500
nmol O 2 , min-I. (mg prot)-l
400
Uninhibited
300
-~ Q
00
'-'
-:.c:
rJi
~ 0
e::1.
§ .::: c..
~
§ to>
0 '"
315
HNO
N
200
eN-resistant N
BHAM-resistant
HNO
N
HNO
Mixture of 89.8±7.3 159.7± 14.6 63.2±9.6 152.6±21.0 88.5±5.6 89.9±8.7
100
substrates
0 500
B
400
Table 3: Enzyme activities of the N and the HNO calli after 21 days of culture. N
300 200 100 0 500
~
400 300 200
c
\ \ \-
100
---
Glucose-6-phosphate dehydrogenase (J.!g NADPH.min-l.mg prot-I) Phosphofructokinase (J.!g NADH.min-l.mg proC I) cytochrome c oxidase (k.min-I.mg prot-I) Peroxidase (nkat. mg proC I) Catalase (J.!kat.mg proC I) Total protein content (mg.gDW- I)
HNO
B.50±O.1l
1B.OO±O.04
2.70±O.OB
1.40±O.O2
10.42±O.O2
5.35±O.46
5.B3±O.54
O.47±O.O6
1.43±O.O2
l.O4±O.O4
50.0±U
103.9±2.5
- . - --:t:
0 0
7 14 21 28 Time of culture (days)
Fig. 3: Uninhibited respiration (A) (J.lmoles O 2 ·h-I . g (DW)-I) ofN (e) and HNO (_) calli and respiration in the presence of BHAM (B) and CN (C). Results (± SD) are means of three separate experiments.
In order to measure the electron transport chain capacities, experiments with isolated mitochondria were performed. In isolated mitochondria, when using a mixture of substrates, a similar pattern was observed as in the whole tissue respiration: higher uninhibited respiration, a similar cytochrome pathway and a higher CN-resistant pathway in HNO callus as compared with N callus (Table 2). Table 3 shows some enzyme activities, not only of the respiratory chains (cytochrome c oxidase), but also of several other pathways such as the pentose phosphate pathway (glucose-6-phosphate dehydrogenase), glycolysis (phosphofructokinase), and activities of peroxidase and catalase involved in H 20 2 detoxification. The results showed a higher G6PDH activity and a lower PFK activity in HNO cells as compared with the N cells. Peroxidase and catalase showed also lower activities in HNO cells (Table 3). Cytochrome c oxidase is two times lower in HNO cells than in N cells. Western blotting (in the absence of reducing agents like DTT) of mitochondrial proteins of Nand HNO calli pre-
~.I.It.~ 'I"
N
HNO
Fig. 4: Amount of alternative protein in mitochondria isolated from N and HNO calli, determined by Western blotting. One hundred J.!g protein per lane for both N and HNO cells. sented a band at about 35 kda protein, which corresponds to the alternative oxidase in its reduced form (Fig. 4). Much more alternative oxidase protein appeared to be present in HNO mitochondria compared with N mitochondria.
Discussion
Respiratory characteristics Total respiration and CN-resistant respiration were much higher in HNO callus than in N callus, but BHAM-resistant, cytochromal respiration was the same in the two types of cal-
316
Badia Bisbis, Anneke M. Wagner, Claire Kevers and Thomas Gaspar [ Glycolysis
I..
PFK
Glueose-6-phosph.k
_G6iiiiiiiiDiiiiHi004~ Pentose phosphate .......
Pentosea
pathway
p-r-----~r=~--+_t----------_.srn~~~~_t~--r=====~ co,
~
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"
----t~~
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~m
nK
OAA
tMDH
MAL
Cytosol
"1°1
accumUla.on Lack in catalase and ~roxidase _-::...-"", ~ Lack in ~ tetrapyrroli com ounds
Fig. 5: Scheme illustrating the relationships between the deviated carbon and nitrogen metabolisms and the induction of the alternative oxidase in HNO cells. CIT, citrate; DAO, diamine oxidase; DH, dehydrogenase; e-, electron flux; GABA, y-aminobutyrate; GABA-T, GABA transaminase; GDC, glutamate decarboxylase; GDH, glutamate dehydrogenase; G6DH, glucose-6-phosphate dehydrogenase, GLU, glutamate; KG, a-Ketoglutarate; MAL, malate; MDH, malate dehydrogenase; OM, oxaloacetate; PAO, polyamine oxidase; Pas, polyamines; PEp, phosphoenolpyruvate; PEPC, PEP carboxylase; PFK, phosphofructokinase; SSDH, succinic semialdehyde dehydrogenase; SUC, succinate.
Ius. HNO callus showed a higher CN-resistant respiration than BHAM-resistant respiration. The two pathways coexisted also in N callus but with a low uptake via the CNresistant pathway as compared with the cytochrome pathway. However, it is not possible to determine exact contributions of the two pathways on the basis of these inhibitors studies (Wagner and Krab, 1995). The results presented here would suggest that the difference observed in uninhibited respiration between the two types of callus was mainly due to a difference in the respiration via the CN-resistant pathway. Accordingly, the respiration rates of isolated mitochondria with a mixture of exogenous substrates (succinate, malate and NADH) were higher in mitochondria from HNO cells, with a higher respiration rate via the CNresistant pathway and no difference in BHAM-resistant cytochrome respiration between the N and the HNO callus. The higher respiration rates in HNO callus could be caused by a difference in overall enzyme capacities or a difference in ADP/ATP ratio, controlling glycolytic flux. If cellular respiration is ADP availability dependent, a strong stimulation of respiration by uncoupler can be expected. Indeed, an increase of total and cytochrome mediated respiration was observed in the presence of the uncoupler. This indicates that the cytochrome pathway is not working at full capacity. Still, uncoupled respiration in HNO cells was higher than in N
cells, indicating that enzyme capacities were higher in the HNO cell line. More alternative oxidase protein (35 kda) was present in mitochondria isolated from HNO callus compared with N callus mitochondria. This is in agreement with a higher capacity of this pathway. The activity of the alternative pathway can be regulated by the total amount of protein, but also by the reduction state of the enzyme (Umbach and Siedow, 1996). Cytochrome c oxidase activities, on the contrary, were higher in N callus than in HNO callus, which apparently does not affect cytochromal respiratory rates. Similar results were found by Van Emmerik et al. (1993). The higher glucose-6-phosphate dehydrogenase and the lower phosphofructokinase activities in HNO callus point to a disturbed sugar metabolism preferentially deviated to the pentose phosphate pathway. This results probably in a lower glycolytic pathway of HNO callus with, as consequence, an atypical tricarboxylic acid cycle, which is replenished by anaplerotic pathways such as malate production by the CO 2 fixation via phosphoenolpyruvate carboxylase and succinate production via the aminobutyrate shunt (see Fig. 5) (Bisbis et al., 1997a; Gaspar et al., 1998). It is not clear whether the lower peroxidase and catalase activities in HNO callus resulted in less H 2 0 2 elimination in this callus compared with the N callus. Further investigations on that H 20 2 accumulation in HNO callus must be performed.
Respiratory Pathways in Sugarbeet Calli
Respiratory pathways and growth The HNO callus presents a lower growth and higher respiratory rate, the contrary is true for the N callus. The maximal cytochromal activities of the two rypes of callus are the same; thus, the ATP synthesis could be expected to be similar. Recent work has shown that the HNO callus presented a higher level of ATP compared with its normal counterpart (Poder et al., 1998). The differences in growth rates of the HNO and N cells cannot then be explained only by the differences in respiratory characteristics. An explanation for the lower growth of the HNO callus could be found in the hormonal status and in several disturbed metabolic pathways already studied (Gaspar et al., 1998). Indeed, the HNO callus is characterized by a higher endogenous auxin than its normal counterpart (Bisbis, 1998), a lower ethylene production (Bisbis et al., 1998), and a lower auxin oxidase activiry (Kevers et al., 1999). On the other hand, we have shown that although the HNO callus contains no more chlorophyll and is nonphotosynthetic as compared with the N callus (Bisbis et al., 1994), still about 20 % of the biomass increase derives from CO 2 fixation versus 44 % for the N callus (Bisbis et al., 1997 a). The CO 2 fixation occurred in HNO callus via the phosphoenolpyruvate carboxylase (Bisbis et al., 1997 a). The HNO callus presents a deficiency in tetrapyrrolic compounds, such as peroxidase and catalase (Bisbis et al., 1998). Some or all of these parameters could contribute to the lower growth with a reduced post-exponential linear growth phase ofHNO, which could indicate a defective cell expansion (Hagege et al.,
1991).
Conclusions
O 2 uptake by HNO cells is higher than in N cells and is more CN-resistant than respiration in N cells. The alternative oxidase protein was shown to be present in a higher amount in HNO callus than in N callus. The HNO callus was a subject of many studies, which showed many metabolic deviations and suggested that the HNO callus is under permanent stress (Gaspar et al., 1998 and refs. therein). In this respect, the observed increase of alternative oxidase protein is interesting. Plants are able to regulate, in a coordinate fashion, the partitioning of electrons between the cytochrome and the alternative pathways to meet changing metabolic requirements (Vanlerberghe and McIntosh, 1992). Little is known, however, about the functions of the alternative oxidase (AP). Alternative oxidase is induced in response to stress conditions and it has been suggested that the alternative oxidase plays a role in the antioxidant defence in plants (Purvis and Shewfelt, 1993; Wagner and Krab, 1995; Wagner and Moore,
1997).
A reaction to the lowered cytochrome c oxidase activiry shown in HNO cells might be the synthesis of extra-alternative oxidase protein, as observed before (Vanlerberghe and McIntosh, 1992; Wagner and Wagner, 1997). It is not clear yet how the signal from the cytochrome pathway to nuclear gene expression is transduced, but reactive oxygen species such as H 20 2 can act as inducers (Wagner, 1995). It is well known that HNO callus synthesizes a higher amount of poly-
317
amines (Hagege et al., 1990) yielding H 20 2 via their degradation. The lower catalase and peroxidase activities could facilitate the accumulation of H 20 2 • An increased alternative oxidase is in accordance with this assumption. Figure 5 summarizes the different metabolic pathways investigated in HNO callus. Acknowledgements
B. Bisbis would like to thank the FNRS (Fonds National de la Recherche Scientifique) for supporting this work, and the scientific and technical staff of IMBW, Molecular Cell Physiology Laboratory.
References
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